Highly-purified nicotinic chloinergic membranes from Torpedo electric tissue contain, in addition to the polypeptides of the acetylcholine receptor, two major proteins of 43,000 and 90,000 daltons. The latter is thought to be a contaminant. The 43,000 dalton protein (43K protein), however, remains associated with the receptor-rich membranes throughout several purification steps but can be removed by procedures that solubilize peripheral membrane proteins. It appears to be located on the cytoplasmic side of the membrane. Receptor function is unaffected by removal of the 43K protein from the membranes but some aspects of the membrane organization change. We have found by immunofluorescence techniques that the 43K protein is located in the inervated membarnes of Torpedo electrocytes and that immunologically-related component is highly- concentrated at the mammalian neuromuscular junction. We propose to investigate the function of the 43K protein in mammalian muscle and Torpedo electric tissue. First, a biochemical comparison of the protein from both tissues will be conducted. We will also attempt to reconstitute the 43K protein with stripped Torpedo membranes to examine its effects on receptor stability. More detailed analysis of the synaptic location of the 43K protein will be carried out by immunoelectron-microscopy and by chemical crosslinking studies. The latter will provide information concerning possible interactions of the 43K protein and the receptor. During synaptogenesis in vivo, the postsynaptic membrane of mamalian muscle undergoes a complex series of changes, many of which involve the acetycholine receptor. We will determine when the 43K protein becomes a part of the postsynaptic membrane, using immunofluorescense and a radioimmune assay, in an attempt to correlate its appearance with one of these developmental steps. The influence of neurons on its induction and distribution will be examined in vitro using co-cultures of nerve and muscle cells. Finally, we will examine neuronal and non-neuronal cells for the presence of 43K protein or components related to it. Since the 43K protein is highly-concentrated in postsynatic membranes, it must play an important role in synaptic structure and/or function. An understanding of its function is necessary before its involvement in pathological states of neuromuscular transmission can be assessed.